Deazaflavin compounds and methods of use thereof

ABSTRACT

The present invention features 5-deazaflavin compounds, pharmaceutical compositions of 5-deazaflavin compounds and methods of treating a patient suffering from cancer, the method comprising administering to a patient one or more 5-deazaflavin compounds of the invention.

The present application claims the benefit of U.S. provisionalapplication No. 60/447,610, filed Feb. 13, 2003, which is incorporatedby reference herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention involves 5-deazaflavin compounds and methods andpharmaceutical compositions that comprise such compounds. Compounds ofthe invention can be effective to regulate p53 and MDM2 stability andactivity as well as to act as therapeutic agents in cancer.

2. Background

The development of cancer can depend on the accumulation of specificgenetic alterations that allow aberrant cell proliferation, includinggrowth of tumor cells. Protection from such aberrant growth is providedby several mechanisms that work by inducing apoptotic cell death incells undergoing oncogenic changes. Therefore, for a tumor cell tosurvive, it must acquire genetic alterations that perturb the linkbetween abnormal growth and cell death. The p53 tumor suppressor proteincan induce apoptotic cell death and plays a pivotal role in tumorsuppression. Wild type p53 functions as a transcriptional regulator tocoordinately control multiple pathways in cell cycling, apoptosis, andangiogenesis.

Loss of the ability to induce p53 or other loss of p53 activity can leadto uncontrolled cell proliferation of the affected cells and tumorgrowth. In approximately 50% of human cancers, a wild type p53 gene isnevertheless retained. In such cancers, the defect that frequentlyoccurs is a failure to stabilize and activate p53 to thereby preventtumor development.

The MDM2 protein plays an important role in targeting the degradation ofp53 in normal cells to allow normal growth and development. Inparticular, inhibition of MDM2 is required to allow activation of a p53response. In tumors with wild type p53, defects can occur that lead toincreased MDM2 activity, whereby p53 function cannot be induced.

Ubiquitin-mediated proteolysis is an important pathway of non-lysosomalprotein degradation that controls the timed destruction of a number ofcellular regulatory proteins including p53. See Pagano, 1997 FASEB J.11:1067. Ubiquitin is an evolutionary highly conserved 76-amino acidpolypeptide which is abundantly present in eukaryotic cells. Theubiquitin pathway leads to the covalent attachment of a poly-ubiquitinchain to target substrates which are then degraded by a multi-catalyticproteasome complex.

A number of the steps of regulating protein ubiquitination are known. Inparticular, initially the ubiquitin activating enzyme (E1) forms a highenergy thioester linkage with ubiquitin. Ubiquitin is then transferredto a reactive cysteine residue of one of many ubiquitin conjugatingenzymes known as Ubc or ubiquitin E2 enzymes. The final transfer ofubiquitin to a target protein involves one of many ubiquitin proteinligases (E3s). MDM2 is such a ubiquitin ligase that mediates thetransfer of ubiquitin to p53.

It thus would be desirable to have new compounds that have use intreatment of undesired cell proliferation, including in treatmentagainst cancer cells. It would be particularly desirable to have newcompounds that could modulate or stabilize p53 activity by inhibitingMDM2-mediated ubiquitination.

SUMMARY OF THE INVENTION

The invention involves a family of 7-nitro-5-deazaflavin compounds.Compounds of the invention may be useful as anti-cancer agents. Indeed,we have found that 7-nitro-5-deazaflavin compounds can stabilize p53 inmammalian cells. Preferred 7-nitro-5-deazaflavin compounds additionallyinhibit MDM2 activity. See for instance, the results set forth in theExamples, which follow.

More particularly, the invention provides compounds of the followingFormula I:

wherein:

Ar is a monosubstituted carbocyclic aryl group;

R¹ is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclic aryl, optionallysubstituted heteroaryl, optionally substituted cycloalkyl, optionallysubstituted heteroalicyclic, or optionally substituted aminoalkyl;

R² and R³ are independently selected from the group consisting ofhydrogen, amino, hydroxy, cyano, nitro, carboxylate, carboxamide,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclic aryl, optionallysubstituted heteroaryl, optionally substituted alkoxy, optionallysubstituted mono- or di-alkyl amino, optionally substituted cycloalkyl,optionally substituted heteroalicyclic, or optionally substitutedaminoalkyl; and

n is an integer from 0 to 3; and pharmaceutically acceptable saltsthereof.

These compounds exhibit detectable inhibition of MDM2 E3 ubiquitinligase activity in an in vitro assay (defined herein as a “standard MDM2activity in vitro assay”), particularly a detectable decrease in MDM2ubiquitination as measured by a decrease in the addition of ubiquitinmolecules to MDM2 as assessed using an SDS-PAGE gel based means ofassessment. See the assay of Example 2, which follows. The protocol ofthat assay of Example 2 is defined herein to be a “standard MDM2activity in vitro assay”.

Particularly preferred compounds of the invention also may be selectivefor cancer cells relative to normal cells of a subject, i.e., suchpreferred compounds will exhibit reduced cell death in normal cellsrelative to targeted cancer cells. In particular, such preferredcompounds can inhibit proliferation or induce apoptosis of targetedcancer cells, without exerting significant toxicity to normal(non-cancer) cells that may be contacted with the administeredcompound(s).

Compounds of the invention are useful for a number of therapeuticapplications. In particular, the invention includes methods fortreatment and prophylaxis of cancer, including cancers of the breast,lung, prostate, brain, liver, testes, skin, among others. Disseminatedcancers (e.g., leukemias) as well as solid tumors may be treated bymethods of the invention. Treatment methods of the invention may includeadministration of an effective amount of one or more compounds of theinvention to cancer cells, such as those mentioned above. Moreparticular methods include administering an effective amount of acompound of the invention to a subject such as a mammal, particularly aprimate, e.g., a human that is suffering from or susceptible to(prophylactic treatment) abnormal cell proliferation, especially acancer, such as a cancer mentioned above. Preferably, a subject isidentified and selected that is susceptible or suffering undesired cellgrowth, especially cancer, such as a cancer mentioned above. Aneffective amount of one or more compounds of the invention suitably isan amount of one or more of the compounds of the invention sufficient tostabilize p53 in cells. The invention also includes use of one or morecompounds disclosed herein, in combination or coordination with existingchemotherapies and/or radiotherapeutic protocols.

The invention also includes methods to stabilize p53 in cells,particularly mammalian cells, such as primate cells especially humancells.

Preferred methods of the invention are suitable for use in tumor growthregulation and comprise the administration of compounds of Formula I totargeted cells.

In a further aspect, the invention provides use of a compound ofFormulae I, II, III and/or IV as defined herein, for the treatment orprevention (including prophylactic treatment) of a disease or conditionas disclosed herein, including treatment or prevention of cancer orother undesired cell growth or proliferation.

In a yet further aspect, the invention provides use of a compound ofFormulae I, II, III and/or IV as defined herein, for the preparation ofa medicament for the treatment or prevention (including prophylactictreatment) of a disease or condition as disclosed herein, includingtreatment or prevention of cancer or other undesired cell growth orproliferation.

Pharmaceutical compositions also are provided which comprise a compoundof Formulae I, II, III or IV as defined herein, optionally incombination with a pharmaceutically acceptable carrier. Preferably, suchpharmaceutical compositions are packaged together with instructions(written) for use of the compounds for a therapeutic application,particularly to treat a subject for undesired cell growth, such as acancer identified above.

The invention further provides methods for identifying (e.g., throughscreening) other compounds possessing activity as anti-cancer agents.The assays are preferably based on measurement of inhibition of MDM2ubiquitin ligase activity, such as by standard MDM2 activity in vitroassay. Potential inhibitors of MDM2 would regulate the stability andfunction of p53 and MDM2. Preferably the assays measure theself-ubiquitylation of MDM2 in the presence of candidate compounds. Anincreased inhibition of the self-ubiquitylation of MDM2 in the presenceof candidate compounds, as compared to control samples is indicative ofa potential anti-tumor compound. Preferably, a candidate compoundsinhibits self-ubiquitylation of MDM2 by at least 20% greater as comparedto a control (no candidate compound administered) as measured in astandard MDM2 activity in vitro assay, more preferably a candidatecompound inhibits self-ubiquitylation of MDM2 by at least about 30%,40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to a control system (nocandidate compound adrninistered) as measured in a standard MDM2activity in vitro assay. Additional in vitro assays of use inidentifying agents include inhibition of p53 ubiquitination by MDM2(described infra). In such an assay, p53 produced in human or mousecells or translated in a cell free eukaryotic expression system ispre-bound to MDM2 and inhibition of p53 ubiquitination is assessed.

In another preferred embodiment, potential inhibitors of MDM2 thatregulate the stability and function of p53, can be determined in a cellbased assay. Potential inhibitors of MDM2 would regulate the stabilityand function of p53 and MDM2. Preferably, the assays measure number ofcells undergoing apoptosis due to the inhibition of MDM2 induced p53degradation in tumor cells in the presence and/or absence of candidatecompounds as compared to normal cells in the presence and/or absence ofcandidate compounds. The assay can also measure stabilization of p53 andMDM2 in cells following treatment with one or more candidate compounds.

In such assays of the invention, an increase in the number of cellsundergoing apoptosis in the presence of candidate compounds in tumorcells, as compared to normal untreated cells is indicative of apotential anti-tumor compound. Preferably a candidate compound increasesapoptosis of tumor cells by at least 20% as compared to a control system(no candidate compound administered), more preferably a candidatecompound increases apoptosis of a tumor cell by at least about 30%, 40%,50%, 60%, 70%, 80%, 90% or 100% as compared to a control system (nocandidate compound administered). That is, for example, 80% increase ofapoptosis refers to number of cells still surviving as compared to thecontrols. Apoptosis is preferably measured by visual observation (e.g.,blebbing or trypan blue retention). Nucleic acids from cells havingundergone apoptosis can be run on gels showing the characteristic 200 bpnucleic acid ladder that is indicative of cells having undergoneapoptosis, or cells with less than a G1 DNA content can be identified byfluorescence activated cell sorting. Other assays for apoptosis includeTUNEL assays or detection of caspase activation.

Assays of the invention also are useful for assessing MDM2 inhibition isin in vitro and in vivo systems.

In another aspect, the invention includes compounds that can interactwith E1 and/or E2 enzymes. Compounds that inhibit at E1 and/or E2 levelswould be useful drug candidates that could indirectly interfere with theactivity of ubiquitin ligases, particularly MDM2. Since interactionsbetween E1 and E2 share similarities to those between E2 and E3,compounds of the invention may also inhibit loading of E2 by E1, andthereby, inhibit the activity of ubiquitin ligases such as MDM2.

Other aspects of the invention are described infra.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a graph showing that MDM2 exhibited a signal to backgroundratio of ˜100 in self-ubiquitylation assay. Time course of MDM2self-ubiquitylation revealed linear reaction kinetics and no benefit ofshaking within the first 60 min. Samples were incubated at roomtemperature and not shaken for ease of automation as shown in FIG. 1B.FIG. 1C is a graph showing that the MDM2 assay signal is a function oftime between addition of the antibody and measuring in an M8 analyzer.

FIGS. 2A-2G are gels showing the results obtained using in vitro gelassays. FIGS. 2A-2C shows the ubiquitination of MDM2 and Nedd4 in thepresence or absence of candidate compounds.

FIG. 2A is a gel showing the inhibition of MDM2 ubiquitination in thepresence or absence of candidate compounds. Using the ARF peptide as apositive control for inhibition of MDM2 ubiquitination, these assaysidentified four compounds that showed an ability to significantlyinhibit MDM2 E3 ligase (FIG. 2A lanes 7, 9, 10, 11).

FIG. 2B is a gel showing typical results obtained in a screen todetermine whether the compounds were selective in their ability toinhibit MDM2 as compared to their effect on the activity of anotherunrelated E3 ligase, Nedd4.

FIG. 2C is gel showing the results of an independent experiment furtherdemonstrating the specificity of MDM2 by 98C07(10-(-3-chloro-phenyl)-7-nitro-10H-pyrimido [4,5-b]quinoline-2,4-dione)(lane 2).

FIGS. 2D and 2E show the effect of compounds on the more proximal stepsin the ubiquitination process, formation of thiol-ester linkages with E1(FIG. 2D) and with E2 (FIG. 2E).

FIG. 2F shows inhibition of MDM2 auto-ubiquitination by two other closefamily members of 98C07.

FIG. 2G shows the results obtained from two compounds that inhibit p53ubiquitination by MDM2. 98C07 and 98D07(10-(-4-chloro-phenyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-2,4-dione)both exhibit significant dose-dependent inhibition of p53 ubiquitinationafter cellular p53 is pre-bound to GST-MDM2.

FIGS. 3A-3C show the results from the in vivo cell based assay foraccumulation of MDM2 and p53. Normal human fibroblast MRC-5 were chosento determine whether the compounds from the high throughput screeningcan inhibit the E3 activity of MDM2, i.e. stabilizing MDM2 and p53 invivo. Compounds referred to as 98C07, 98D07, and 98E07(10-(-4-methyl-phenyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-2,4-dione)were all found to specifically increase MDM2 and p53 compared to aseries of other compounds identified in the high throughput screen andwere not further considered (FIG. 3A and FIG. 3B). As shown in FIG. 3B,they all increased MDM2 and p53 levels in MRC-5 cells. The specificityof the compounds was also revealed by examining whether they affect thelevels of the HECT domain E3 Nedd4 and p27, which is ubiquitinated by aRING finger-dependent SCF E3. As shown in FIG. 3B, the amounts of bothNedd4 and p27 were not changed significantly by any of the 98 familycompounds. Moreover, while adriamycin increased only p53 and proteosomeinhibitor (LLNL) accumulated MDM2, p53, and p21, these compounds onlyincreased the amount of MDM2 and p53, but not p21, indicating they arespecific for the E3 activity of MDM2 (FIG. 3C).

FIG. 4 shows a graph (left panel) and two gels (right panel)illustrating that the compounds stabilize p53 in untransformed cells(retinal pigment epithelial cells [RPE]) as well as transformed cells(RPE/E1A). However, only the transformed cells are sensitive to p53 andkilled by the compounds.

FIGS. 5A-5B are graphs showing results from an evaluation as to whetherapoptosis stimulated by the compounds requires p53 expression. E1A andRas transformed MEFs from cells either expressing p53 (C8) or p53^(−/−)(A9) cells were compared in their capacity to activate effector caspases(FIG. 5 a) and cell death (FIG. 5 b).

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, we now provide new 7-nitro-5-deazaflavin compoundswhich can be useful in cancer therapies.

Preferred compounds of Formula I of the invention include those of thefollowing Formula II:

wherein:

R¹ is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclic aryl, optionallysubstituted heteroaryl, optionally substituted cycloalkyl, optionallysubstituted heteroalicyclic, or optionally substituted aminoalkyl;

R² and R³ are independently selected from the group consisting ofhydrogen, amino, hydroxy, cyano, nitro, carboxylate, carboxamide,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclic aryl, optionallysubstituted heteroaryl, optionally substituted alkoxy, optionallysubstituted mono- or di-alkyl amino, optionally substituted cycloalkyl,optionally substituted heteroalicyclic, or optionally substitutedaminoalkyl;

R⁴ is selected from the group consisting of amino, halogen, hydroxy,cyano, nitro, carboxylate, carboxamide, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted alkoxy, optionally substituted mono- or di-alkylamino, optionally substituted cycloalkyl, optionally substitutedheteroalicyclic, or optionally substituted aminoalkyl; and

n is an integer from 0 to 3; and pharmaceutically acceptable saltsthereof.

Particularly preferred compounds of Formula II provided by the inventioninclude those compounds in which

R¹ is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₂₋₆alkenyl, C₂₋₆alkynyl, C₁₋₆hydroxyalkyl, C₁₋₆aminoalkyl, C₆₋₁₂aryl,C₃₋₁₂heteroaryl having between 1 and 4 ring heteroatoms, C₇₋₁₂aralkyl,C₃₋₁₂cycloalkyl, and C₃₋₁₂cycloheteroalkyl;

R² and R³ are independently selected from the group consisting ofhydrogen, amino, hydroxy, cyano, nitro, C₁₋₆alkyl, C₂₋₆alkenyl,C₂₋₆alkynyl, C₁₋₆alkoxy, C₁₋₆hydroxyalkyl, C₁₋₆aminoalkyl, C₆₋₁₂aryl,C₃₋₁₂heteroaryl having between 1 and 4 ring heteroatoms, C₇₋₁₂aralkyl,C₃₋₁₂cycloalkyl, C₃₋₁₂cycloheteroalkyl, mono or di (C₁₋₆alkyl)amino, orcarboxylate;

R⁴ is selected from the group consisting of amino, halogen, hydroxy,C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, C₁₋₆alkoxy, or mono or di(C₁₋₆alkyl)amino; and

n is an integer from 0 to 3; and pharmaceutically acceptable saltsthereof.

Other preferred compounds according to Formula I or Formula II includethose represented by the following Formula III:

R¹ is selected from the group consisting of hydrogen, C₁₋₆alkyl,C₂₋₆alkenyl, C₂₋₆alkynyl, C₁₋₆hydroxyalkyl, C₁₋₆aminoalkyl, C₆₋₁₂aryl,C₃₋₁₂heteroaryl having between 1 and 4 ring heteroatoms, C₇₋₁₂aralkyl,C₃₋₁₂cycloalkyl, and C₃₋₁₂cycloheteroalkyl;

R² is selected from the group consisting of hydrogen, amino, hydroxy,cyano, nitro, C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, C₁₋₆alkoxy,C₁₋₆hydroxyalkyl, C₁₋₆aminoalkyl, C₆₋₁₂aryl, C₃₋₁₂heteroaryl havingbetween 1 and 4 ring heteroatoms, C₇₋₁₂aralkyl, C₃₋₁₂cycloalkyl,C₃₋₁₂cycloheteroalkyl, mono or di (C₁₋₆alkyl)amino, or carboxylate;

R⁴ is selected from the group consisting of amino, halogen, hydroxy,C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, C₁₋₆alkoxy, or mono or di(C₁₋₆alkyl)amino; and

n is an integer from 0 to 3; and pharmaceutically acceptable saltsthereof.

Particularly preferred compounds of any one of Formula I, II, or IIIprovided by the invention include those compounds wherein R¹, R², andeach occurrence of R³ are selected from the group consisting of hydrogenand C₁₋₆alkyl; and R⁴ is selected from the group consisting of chloro,fluoro, bromo, methyl, ethyl, hydroxy, and methoxy.

Additionally preferred compounds of the present invention include thosecompounds of the following Formula IV:

wherein R⁴ is selected from the group consisting of amino, halogen,hydroxy, C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, C₁₋₆alkoxy, or mono or di(C₁₋₆alkyl)amino; and pharmaceutically acceptable salts thereof.

Particularly preferred compounds according to Formula IV include thosewherein R⁴ is selected from the group consisting of a chloro, fluoro, ormethyl group, and R⁴ is attached to the 3 or 4 position of the phenylring.

Other particularly preferred compounds provided by the invention includethe following, where the compound structure is depicted directly abovethe compound name.

10-(3-chloro-phenyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-2,4-dione,

10-(4-chloro-phenyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-2,4-dione, and

10-(4-methyl-phenyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-2,4-dione.

Suitable alkyl substituent groups of compounds of the invention (whichincludes compounds of Formulae I, II, III and IV as defined above)typically have from 1 to about 12 carbon atoms, more preferably 1 toabout 8 carbon atoms, still more preferably 1, 2, 3, 4, 5, or 6 carbonatoms. As used herein, the term alkyl unless otherwise modified refersto both cyclic and noncyclic groups, although of course cyclic groupswill comprise at least three carbon ring members. Preferred alkenyl andalkynyl groups of compounds of the invention have one or moreunsaturated linkages and typically from 2 to about 12 carbon atoms, morepreferably 2 to about 8 carbon atoms, still more preferably 2, 3, 4, 5,or 6 carbon atoms. The terms alkenyl and alkynyl as used herein refer toboth cyclic and noncyclic groups, although straight or branchednoncyclic groups are generally more preferred. Preferred alkoxy groupsof compounds of the invention include groups having one or more oxygenlinkages and from 1 to about 12 carbon atoms, more preferably from 1 toabout 8 carbon atoms, and still more preferably 1, 2, 3, 4, 5 or 6carbon atoms. Preferred alkylthio groups of compounds of the inventioninclude those groups having one or more thioether linkages and from 1 toabout 12 carbon atoms, more preferably from 1 to about 8 carbon atoms,and still more preferably 1, 2, 3, 4, 5, or 6 carbon atoms. Preferredalkylsulfinyl groups of compounds of the invention include those groupshaving one or more sulfoxide (SO) groups and from 1 to about 12 carbonatoms, more preferably from 1 to about 8 carbon atoms, and still morepreferably 1, 2, 3, 4, 5, or 6 carbon atoms. Preferred alkylsulfonylgroups of compounds of the invention include those groups having one ormore sulfonyl (SO₂) groups and from 1 to about 12 carbon atoms, morepreferably from 1 to about 8 carbon atoms, and still more preferably 1,2, 3, 4, 5 or 6 carbon atoms. Preferred aminoalkyl groups include thosegroups having one or more primary, secondary and/or tertiary aminegroups, and from 1 to about 12 carbon atoms, more preferably 1 to about8 carbon atoms, still more preferably 1, 2, 3, 4, 5, or 6 carbon atoms.Secondary and tertiary amine groups are generally more preferred thanprimary amine moieties. Suitable heteroaromatic groups of compounds ofthe invention contain one or more N, O or S atoms and include, e.g.,coumarinyl including 8-coumarinyl, quinolinyl including 8-quinolinyl,pyridyl, pyrazinyl, pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl,oxazolyl, oxidizolyl, triazole, imidazolyl, indolyl, benzofuranyl andbenzothiazole. Suitable heteroalicyclic groups of compounds of theinvention contain one or more N, O or S atoms and include, e.g.,tetrahydrofuranyl, thienyl, tetrahydropyranyl, piperidinyl, morpholinoand pyrrolidinyl groups. Suitable carbocyclic aryl groups of compoundsof the invention include single and multiple ring compounds, includingmultiple ring compounds that contain separate and/or fused aryl groups.Typical carbocyclic aryl groups of compounds of the invention contain 1to 3 separate or fused rings and from 6 to about 18 carbon ring atoms.Specifically preferred carbocyclic aryl groups include phenyl; naphthylincluding 1-naphthyl and 2-naphthyl; biphenyl; phenanthryl; anthracyl;and acenaphthyl. Substituted carbocyclic groups are particularlysuitable including substituted phenyl, such as 2-substituted phenyl,3-substituted phenyl, 4-substituted phenyl, 2,3-substituted phenyl,2,4-substituted phenyl, and 2,4-substituted phenyl; and substitutednaphthyl, including naphthyl substituted at the 5, 6 and/or 7 positions.

Suitable aralkyl groups of compounds of the invention include single andmultiple ring compounds, including multiple ring compounds that containseparate and/or fused aryl groups. Typical aralkyl groups contain 1 to 3separate or fused rings and from 6 to about 18 carbon ring atoms.Preferred aralkyl groups include benzyl and methylenenaphthyl(—CH₂-naphthyl), and other carbocyclic aralkyl groups, as discussedabove.

Suitable heteroaralkyl groups of compounds of the invention includesingle and multiple ring compounds, including multiple ring compoundsthat contain separate and/or fused heteroaromatic groups, where suchgroups are substituted onto an alkyl linkage. More preferably, aheteroaralkyl group contains a heteroaromatic group that has 1 to 3rings, 3 to 8 ring members in each ring and from 1 to 3 hetero (N, O orS) atoms, substituted onto an alkyl linkage. Suitable heteroaromaticgroups substituted onto an alkyl linkage include, e.g., coumarinylincluding 8-coumarinyl, quinolinyl including 8-quinolinyl, pyridyl,pyrazinyl, pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl,oxidizolyl, triazole, imidazolyl, indolyl, benzofuranyl andbenzothiazole.

Suitable heteroalicyclicalkyl groups of compounds of the inventioninclude single and multiple ring compounds, where such groups aresubstituted onto an alkyl linkage. More preferably, aheteroalicylicalkyl group contains at least one ring that has 3 to 8ring members from 1 to 3 hetero (N, O or S) atoms, substituted onto analkyl linkage. Suitable heteroalicyclic groups substituted onto an alkyllinkage include, e.g. tetrahydrofuranyl, thienyl, tetrahydropyranyl,piperidinyl, morpholino and pyrrolidinyl groups.

As discussed above, various substituent groups (R², R³, and R⁴) ofFormulae I through IV may be optionally substituted. A “substituted” R¹,R², R³, and R⁴ group or other substituent may be substituted by otherthan hydrogen at one or more available positions, typically 1 to 3 or 4positions, by one or more suitable groups such as those disclosedherein. Suitable groups that may be present on a “substituted” R¹, R²,R³, and R⁴ group or other substituent include e.g. halogen such asfluoro, chloro, bromo and iodo; cyano; hydroxyl; nitro; azido; alkanoylsuch as a C₁₋₆ alkanoyl group such as acyl and the like; carboxamido;alkyl groups including those groups having 1 to about 12 carbon atoms,or 1, 2, 3, 4, 5, or 6 carbon atoms; alkenyl and alkynyl groupsincluding groups having one or more unsaturated linkages and from 2 toabout 12 carbon, or 2, 3, 4, 5 or 6 carbon atoms; alkoxy groups havingthose having one or more oxygen linkages and from 1 to about 12 carbonatoms, or 1, 2, 3, 4, 5 or 6 carbon atoms; aryloxy such as phenoxy;alkylthio groups including those moieties having one or more thioetherlinkages and from 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5 or 6carbon atoms; alkylsulfinyl groups including those moieties having oneor more sulfinyl linkages and from 1 to about 12 carbon atoms, or 1, 2,3, 4, 5, or 6 carbon atoms; alkylsulfonyl groups including thosemoieties having one or more sulfonyl linkages and from 1 to about 12carbon atoms, or 1, 2, 3, 4, 5, or 6 carbon atoms; aminoalkyl groupssuch as groups having one or more N atoms and from 1 to about 12 carbonatoms, or 1, 2, 3, 4, 5 or 6 carbon atoms; carbocyclic aryl having 6 ormore carbons, particularly phenyl (e.g. an R group being a substitutedor unsubstituted biphenyl moiety); aralkyl having 1 to 3 separate orfused rings and from 6 to about 18 carbon ring atoms, with benzyl beinga preferred group; aralkoxy having 1 to 3 separate or fused rings andfrom 6 to about 18 carbon ring atoms, with O-benzyl being a preferredgroup; or a heteroaromatic or heteroalicyclic group having 1 to 3separate or fused rings with 3 to about 8 members per ring and one ormore N, O or S atoms, e.g. coumarinyl, quinolinyl, pyridyl, pyrazinyl,pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl,indolyl, benzofuranyl, benzothiazolyl, tetrahydrofaranyl,tetrahydropyranyl, piperidinyl, morpholino and pyrrolidinyl.

Compounds of the invention may exist in differing isomeric forms,including as differing stereoisomers, geometric isomers and the like.Compounds suitable for use in the methods of the present inventioninclude any and all different single pure isomers and mixtures of two ormore isomers. The term isomers is intended to include diastereoisomers,enantiomers, regioisomers, structural isomers, rotational isomers,tautomers, and the like. For compounds which contain one or morestereogenic centers, e.g., chiral compounds, the methods of theinvention may be carried out with a enantiomerically enriched compound,a racemate, or a mixture of diastereomers. Preferred enantiomericallyenriched compounds have an enantiomeric excess of 50% or more, morepreferably the compound has an enantiomeric excess of 60%, 70%, 80%,90%, 95%, 98%, or 99% or more. In preferred embodiments, only oneenantiomer or diastereomer of a chiral 7-nitro-5-deazaflavin compound isadministered to cells or a subject.

Compounds of the invention can be readily prepared by known syntheticmethods. For example, a compound of Formula I may be prepared bycyclocondensation of a 6-(N-arylamino)uracil and a2-halo-5-nitro-benzaldehyde to form 10-aryl-7-nitro-5-deazaflavincompounds. In other embodiments, various compounds of the invention canbe readily prepared having a variety of functionalized aryl groups bysynthesizing 7-nitro-5-deazaflavin compounds which are unsubstituted atthe 10 position from 6-aminouracil and a 2-halo-5-nitro-benzaldehydecompound. Subsequent arylation at the 10 position may be carried out inany convenient manner such as palladium/phosphine catalyzed arylation ofthe 7-nitro-5-deazaflavin compound with an activated aryl reagent suchas an aryl bromide, aryl iodide, aryl boronic acid, aryl tin reagent,aryl silane, or the like.

As discussed above, it has been found that 7-nitro-5-deazaflavincompounds of the present invention including those compounds representedby any one of Formula I-IV are capable of stabilizing p53. Although notbeing bound by any theory, it is believed that preferred compounds ofthe invention can stabilize p53 activity in transformed cells byinhibition of MDM2 ubiquitin ligase activity. More particularly, it isbelieved compounds of the invention, including those compounds ofFormula I-IV, are capable of inhibiting the ubiquitin ligase (E3)activity of MDM2.

As discussed above, the invention includes methods for treating orpreventing (prophylactic treatment) against undesired cell growth orproliferation.

Preferred therapeutic methods of the invention include treatingmalignancies, including solid tumors and disseminated cancers. Exemplarytumors that may be treated in accordance with the invention include e.g.cancers of the lung, prostate, breast, liver, colon, breast, kidney,pancreas, brain, skin including malignant melanoma and Kaposi's sarcoma,testes or ovaries, or leukemias or lymphomia including Hodgkin'sdisease.

The therapeutic methods of the invention generally compriseadministration of an effective amount of one or more compounds of theinvention to a subject including a mammal, such as a primate, especiallya human, in need of such treatment.

The treatment methods of the invention also will be useful for treatmentof mammals other than humans, including for veterinary applications suchas to treat horses and livestock e.g. cattle, sheep, cows, goats, swineand the like, and pets (companion animals) such as dogs and cats.

For diagnostic or research applications, a wide variety of mammals willbe suitable subjects including rodents (e.g. mice, rats, hamsters),rabbits, primates and swine such as inbred pigs and the like.Additionally, for in vitro applications, such as in vitro diagnostic andresearch applications, body fluids (e.g., blood, plasma, serum, cellularinterstitial fluid, saliva, feces and urine) and cell and tissue samplesof the above subjects will be suitable for use.

Compounds of the invention may be administered singularly (i.e., soletherapeutic agent of a regime) to treat or prevent diseases andconditions such as undesired cell proliferation as disclosed herein.

Compounds of the invention also may be administered as a “cocktail”formulation, i.e., coordinated administration of one or more compoundsof the invention together with one or more other active therapeutics.For instance, one or more compounds of the invention may be administeredin coordination with a regime of one or more other chemotherapeuticagents, particularly a compound that functions against cancer cellsother than by p53 stabilization such as an antineoplastic drug, e.g., analkylating agent (e.g., mechloroethamine, chlorambucil, cyclophosamide,melphalan, or ifosfamide), an antimetabolite such as a folate antagonist(e.g., methotrexate), a purine antagonist (e.g. 6-mercaptopurine) or apyrimidine antagonist (e.g., 5-fluorouracil). Other, non-limitingexamples of chemotherapeutic agents that might be used in coordinationwith one or more compounds of the invention include taxanes andtopoisomerase inhibitors. In addition, other non-limiting examples ofactive therapeutics include biological agents, such as monoclonalantibodies or IgG chimeric molecules, that achieve their therapeuticeffect by specifically binding to a receptor or ligand in a signaltransduction pathway associated with cancer.

A particularly suitable combination protocol may include coordinatedadministration of one or more compounds of the invention with a compoundthat can activate but not necessarily stabilize p53, e.g. a therapeuticagent that can enhance interaction of p53 with histone acetylases.

Compounds of the invention can be administered by a variety of routes,such as orally or by injection, e.g., intramuscular, intraperitoneal,subcutaneous or intravenous injection, or topically such astransdermally, vaginally and the like.

In a most preferred embodiment, the compounds of the invention areadministered intravenously. Compounds of the invention may be suitablyadministered to a subject in the protonated and water-soluble form,e.g., as a pharmaceutically acceptable salt of an organic or inorganicacid, e.g., hydrochloride, sulfate, hemi-sulfate, phosphate, nitrate,acetate, oxalate, citrate, maleate, mesylate, etc. If the compound hasan acidic group, e.g. a carboxy group, base additional salts may beprepared. Lists of additional suitable salts may be found, e.g., inRemington's Pharmaceutical Sciences, 17^(th) ed., Mack PublishingCompany, Easton, Pa.

Compounds of the invention can be employed, either alone or incombination with one or more other therapeutic agents as discussedabove, as a pharmaceutical composition in mixture with conventionalexcipient, i.e., pharmaceutically acceptable organic or inorganiccarrier substances suitable for oral, parenteral, enteral or topicalapplication which do not deleteriously react with the active compoundsand are not deleterious to the recipient thereof. Suitablepharmaceutically acceptable carriers include but are not limited towater, salt solutions, alcohol, vegetable oils, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like which do not deleteriously react withthe active compounds.

Pharmaceutical compositions of the invention include a compound of theinvention packaged together with instructions (written) for therapeuticuse of the compound, particularly to treat a subject suffering from orsusceptible to cancer. Most preferred method of treating the patientwith the pharmaceutical compositions of the invention, is administrationof the compositions intravenously. However, other routes ofadministration of the pharmaceutical compositions can be used.

For oral administration, pharmaceutical compositions containing one ormore compounds of the invention may be formulated as e.g. tablets,troches, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsions, hard or soft capsules, syrups, elixers and thelike. Typically suitable are tablets, dragees or capsules having talcand/or carbohydrate carrier binder or the like, the carrier preferablybeing lactose and/or corn starch and/or potato starch. A syrup, elixiror the like can be used wherein a sweetened vehicle is employed.Sustained release compositions can be formulated including those whereinthe active component is protected with differentially degradablecoatings, e.g., by microencapsulation, multiple coatings, etc.

For parenteral application, e.g., sub-cutaneous, intraperitoneal orintramuscular, particularly suitable are solutions, preferably oily oraqueous solutions as well as suspensions, emulsions, or implants,including suppositories. Ampoules are convenient unit dosages.

The actual amounts of active compounds used in a given therapy will varyaccording to the specific compound being utilized, the particularcompositions formulated, the mode of application, the particular site ofadministration, etc. Optimal administration rates for a given protocolof administration can be readily ascertained by those skilled in the artusing conventional dosage determination tests conducted with regard tothe foregoing guidelines. See also Remington's Pharmaceutical Sciences,supra. In general, a suitable effective dose of one or more compounds ofthe invention, particularly when using the more potent compound(s) ofthe invention, will be in the range of from 0.01 to 100 milligrams perkilogram of bodyweight of recipient per day, preferably in the range offrom 0.01 to 20 milligrams per kilogram bodyweight of recipient per day,more preferably in the range of 0.05 to 4 milligrams per kilogrambodyweight of recipient per day. The desired dose is suitablyadministered once daily, or several sub-doses, e.g. 2 to 4 sub-doses,are administered at appropriate intervals through the day, or otherappropriate schedule. Such sub-doses may be administered as unit dosageforms, e.g., containing from 0.05 to 10 milligrams of compound(s) of theinvention, per unit dosage.

As discussed above, the invention also provides methods (also referredto herein as “screening assays”) for identifying candidate compoundsuseful for treatment against cancer cells or other undesired cellproliferation. Screening assays can be adapted to a high throughputformat to enable the rapid screening of a large number of compounds.Assays and screening methods can be used for identification of compoundspossessing MDM2-specific and/or general inhibition of ubiquitin enzymeinhibitory activity. Thus, in accordance with the invention, methods areprovided to screen candidate compounds which exhibit potentialanti-cancer activity by measuring p53 stability in transformed cellsand/or apoptosis and cell death.

MDM2 protein binds tumor suppressor p53 and targets it forubiquitylation and proteosome-mediated degradation. MDM2 is a RINGfinger-containing E3 ubiquitin ligase for p53. MDM2 also catalyzesself-ubiquitylation, and thus regulates intracellular levels of both p53and itself. Without wishing to be bound by theory, molecules whichinhibit the binding of MDM2 to p53 could be important in identifyingpotential drug compounds that inhibit MDM2 ligase activity that affectsp53 stability. Similarly, interference with the expression of MDM2 by acandidate drug compound can identify anti-tumor compounds that can befurther analyzed using a high-throughput assay described below. As atheoretical illustrative example, expression may be down regulated byadministering small molecules and peptides which specifically inhibitMDM2 expression can also be used.

In theory, such inhibitory molecules can be identified by screening forinterference of the MDM2/p53 interaction where one of the bindingpartners is bound to a solid support and the other partner is labeled.Antibodies specific for epitopes on MDM2 or p53 which are involved inthe binding interaction will interfere with such binding. Solid supportswhich may be used include any polymers which are known to bind proteins.The support may be in the form of a filter, column packing matrix orsephadex beads. Labeling of proteins can be accomplished according tomany techniques. Radiolabels, enzymatic labels, and fluorescent labelscan be used. Alternatively, both MDM2 and p53 may be in solution andbound molecules separated from unbound subsequently. Any separationtechnique may be employed, including immunoprecipitation orimmunoaffinity separation with an antibody specific for the unlabeledbinding partner.

For in vitro assays MDM2 can be expressed as a GST fusion. This allowsfor a high level of expression of protein that can be purified onglutathione Sepharose. Detection of ubiquitination of MDM2 can beaccomplished, for example, using ³²P-labeled ubiquitin, Western blottingwith anti-ubiquitin, or by looking at a shift in the molecular weight ofGST fusion by Western blotting with anti-GST. A variety of in vitroassays that measure levels of self-ubiquitylated MDM2 can be employed,such as for example, immunoprecipitation of ubiquitylated MDM2; gelassays wherein the amount of ubiquitylated MDM2 is measured bydensitometric scanning or where covalent attachment of radio-labeled orotherwise tagged ubiquitin to MDM2 or p53 is measured; Western blotanalysis, or other known techniques such as ELISA, immunoprecipitation,RIA, and the like. Candidate compounds that inhibit self-ubiquitylationof MDM2, as described in detail in the Examples which follow, aredetected by a shift in molecular weight either of MDM2 or of ubiquitinthat becomes covalently attached to MDM2. (See for example Lorrick K L.,et al., Proc. Natl. Acad. Sci. USA, 1999, 96:11364-11369; Fang S., etal., J. Biol. Chem., 2000, 275(12)8945-8951; Ryan K M., et al., Curr.Op. Cell Biol., 2001, 13:332-337; which are herein incorporated byreference in their entirety). MDM2 self-ubiquitylation assays are run(see for example the results shown in FIGS. 2 and 3) in the presence orabsence of a known amount of candidate compound. An aliquot of each ofthe test and control reactions are run on a standard SDS-PAGE gel. Testreactions whereby the candidate compounds inhibit theself-ubiquitylation of MDM2 will have a decrease in high molecularweight ubiquitylated MDM2.

In cellular assays, endogenous or transfected MDM2 is used. Fortransfected MDM2, ubiquitination is evaluated by an upward smear byanti-MDM2 Western blotting after resolution of cell lysates on SDS-PAGE.Alternatively, immunoprecipitation can be accomplished by subjectinglysates from cells (treated and untreated cells) to anti-MDM2, followedby Western Blotting and detecting ubiquitination by using anti-ubiquitinantibodies. Preferred screening methods comprise identifying a candidatecompound based on assessment of p53 stabilization (e.g. half life ofp53) and steady state levels, and the level of MDM2, as compared to acontrol, e.g. normal (non-cancer cells).

Steady-state levels of p53 and MDM2 in the cells can be determined by anumber of approaches. For instance, lysates containing cellular proteincan be immunoprecipitated with, for example, a rabbit anti-p53polyclonal serum or MDM2 polyclonal serum, blotted ontopolyvinylidenedifluoride (PVDF) membranes and probed with a monoclonalantibody cocktail comprising, for example, monoclonal antibodies tovarious epitopes of p53, or MDM2. Such antibodies are commerciallyavailable. Immunoblot analyses of cellular extracts, taken at differenttime points after treatment with a candidate compound is determinativeof the half-life of p53 as compared to normal controls. Thus, increaseor decrease in levels of p53 over periods of time is determinative ofp53 stability based on its half-life and steady state levels. Thelysates can be further purified, for example, by immunoprecipitation ofp53 and/or MDM2 directly or indirectly of MDM2 and p53, or by affinitychromatography. Thus, candidate compounds that inhibit MDM2 ubiquitinligase activity, can be screened for any effect on p53 stability.

Cell-based assays include model systems where primary human epithelialcells (“normal cells”) are compared to the same cells expressing theadenovirus E1A oncogene (“transformed cells”). Activation of p53 was nottoxic to normal cells, but activation of p53 in transformed cellsinduces p53-mediated apoptosis. High concentrations of wild type (wt)p53 protein can induce apoptosis in a variety of different tumor cells.Potential inhibitors of MDM2 would regulate the stability and functionof p53 and MDM2. Preferably the assays measure number of cellsundergoing apoptosis due to MDM2 induced p53 degradation in tumor cellsin the presence or absence of candidate compounds as compared to normalcells in the presence or absence of candidate compounds. An increase inthe number of these cells undergoing apoptosis in the presence ofcandidate compounds in tumor cells, as compared to normal untreatedcells is indicative of a potential anti-tumor compound. Preferably acandidate compound increases apoptosis of tumor cells by at least 20% ascompared to a control (no candidate compound administered), morepreferably a candidate compound increases apoptosis of a tumor cell byat least about 30%. 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared toa control (no candidate compound administered). That is, for example 80%increase of apoptosis refers to a decrease in the numbers of cells stillsurviving as compared to the controls.

Apoptosis can be measured by a variety of techniques. For example,apoptosis can be measured by determination of cell phenotype. Phenotyperefers to how the cell looks, typically microscopically, but gross ormacroscopic appearance can be observed. The phenotype changes dependingon the growth rate of the cells. For instance, the microscopicmorphology of cells that are rapidly dividing and growing is differentthan that of cells undergoing cell death by apoptosis. Determination ofcell phenotype is well within the ability of one of ordinary skill inthe art.

There are also a number of biochemical assays that can be used to detectapoptosis, such as “laddering” of the cellular DNA. When testingcompounds for the ability to induce apoptosis, cell death (notcytostasis) is an endpoint of a compound application to the cell. Aclassic signature of apoptosis is the cleavage of nuclear DNA intonucleosomal subunits. On gels, this gives rise to the appearance of aladder as nucleosomal units are sequentially cleaved from DNA.Observation of a classic DNA ladder is indicative of apoptosis. Forexample, cells are lysed and the high molecular weight DNA is removed bycentrifugation. The aqueous phase is treated with proteinase K to digestproteins. After a phenol/chloroform extraction, the pellet is dissolvedin deionized water and treated with 500 μg/ml RNaseA. The DNA is run ona 2% agarose minigel. Observation for a classic DNA ladder is made and aphotograph can be taken. Cell death is verified by the demonstration ofDNA as represented by the ladder configurations on the gel (see forexample, White E., et al. 1984, J. Virol. 52:410). There are also avariety of other assays available for apoptosis such as “TUNEL” assays(see Gavrieli, Y., et al. (1992) J. Cell. Biol. 119:493).

As discussed above, the invention assays and screening methods foridentification of other compounds possessing anti-cancer activity,including MDM2-specific and/or general inhibition of ubiquitin enzymeinhibitory activity. Thus, in accordance with the invention, methods areprovided to screen candidate compounds which exhibit potentialanti-cancer activity by measuring p53 stability in transformed cellsand/or apoptosis and cell death.

All documents mentioned herein are incorporated herein in their entiretyby reference.

The following non-limiting examples are illustrative of the invention.

EXAMPLE 1 High-Throughput Screening Assay

Method: GST-MDM2 is attached to glutathione-coated paramagnetic beadsand the beads are washed. The beads are mixed with E1, E2 andATP-containing buffer in 10 μl volume per one assay. Enzyme inhibitor isadded in 5 μl. The reaction is started by adding 5 μl of ubiquitin andincubated for 1 hour at room temperature. EDTA and ORI-TAG™ labeledantibody against polyubiquitinated proteins in 130 μl is added andincubated for 1 hour at room temperature. The electrochemiluminesence(ECL)-generated signal is read in a M8 analyzer.

Result: The signal is dramatically reduced when mutant MDM2-H457S isused. XIAP, an unrelated RING-finger E3, demonstrated a 4-fold higheractivity. MDM2 exhibited a signal to background ratio of ˜100 inself-ubiquitylation assay. FIG. 1A is a graph showing that MDM2exhibited a signal to background ratio of ˜100 in self-ubiquitylationassay. Time course of MDM2 self-ubiquitylation revealed linear reactionkinetics and no benefit of shaking within the first 60 min. Samples wereincubated at room temperature and not shaken for ease of automation asshown in FIG. 1B. FIG. 1C is a graph showing that the MDM2 assay signalis a function of time between addition of the antibody and measuring inM-8. The curve demonstrated the MDM2 assay signal as a function of timebetween addition of the antibody and measuring in M-8. The assay isstable overnight at room temperature.

EXAMPLE 2 In vitro Assays for Ubiquitination and Thiol-ester BondFormation

Methods:

a. “Standard MDM2 in vitro Ubiguitination Assay” (E1+E2+E3 Assay)

1 pmole per experimental point of bacterially expressed GST-MDM2 (orGST-Nedd) was coupled to glutathione Sepharose (GS) for 30 minutes atroom temperature with tumbling, followed by 3× wash with 50 mM Tris pH7.5. Following this, 20 μl of 1× buffer was added (40 μl 10× reactionbuffer*, 40 μl 10× PCK**, 320 μl dH₂O). The test compound in DMSO isthen added to the desired concentration with an equal volume of DMSOused as a control. Samples are incubated with shaking for 1 hr at 23° C.To carry out the reaction, a pre-made cocktail of Rabbit E1 (Calbiochem#6620700)/UbcH5B/³²P Ub cocktail (1 μl/0.5 μl/1 μl) is added followed by15 minutes shaking at 30° C. The reaction is terminated by addition of 8μl 4× reducing SDS-PAGE loading buffer. After dissociating proteins fromthe beads at 100° C. for 2 minutes, samples are resolved on 6% PAGEfollowed by exposure of the dried gel to phosphor screen. Note: ³²P Ubis derived from GST-Ub that has been engineered to include a PKAphosphorylation site. This fusion protein is purified on glutathioneSepharose, phosphorylated, following this, the ³²P Ub ubiquitin iscleaved and purified away from the thrombin.

*10× Buffer

-   500 mM Tris (pH 7.5)-   2 mM ATP-   5 mM MgCl₂-   1 mM DTT-   10 mM creatine phosphate (Sigma P4635) (45 mg/10 ml).    **10× PCK-   Sigma C7886, 1000 U, reconstitute in 200 μl 10 mM Tris pH 8.0.

b. “E1 only” Assay.

2 μl rabbit E1 +12 μl of 1× reaction buffer are mixed together with thetest compound followed by addition of 1 μl of ³²P Ub is added for 10minutes at room temperature followed by resolution by SDS-PAGE undernon-reducing conditions to maintain thiol-ester linkages and exposure asabove.

c. E1+E2 Assay with Immobilized E2

20 pmoles of bacterially-expressed GST-UbcH5B is bound to GS for 30minutes at room temperature after washing in 50 mM Tris pH 7.5. Twenty(20) μl of 1× reaction buffer is added. After incubation with the testcompound for 1 hour at 23° C. the beads were washed with 50 mM Tris pH7.5. This was followed by addition of Rabbit E1/32P Ub cocktail (1 μl/1μl) followed by incubation at 1 hour shaking at 23° C. This is followedby resolution by SDS-PAGE under non-reducing conditions and exposure asabove.

d. In Vitro R53 Ubiguitination Assay

p53 protein from SaOS-p53 inducible cell lysate was purified from cellsusing GST-MDM2 (5 pmol) pre-bound to GS beads. Samples were thenincubated with test compounds as above. Subsequently 2 μl rabbit E1, 1μl UbcH5b, and 10 μg of ubiquitin were added. After reaction for 15 minat 23° C., samples were subject to SDS-PAGE under reducing conditions,transferred to nitrocellulose membranes and immunoblotted with anti-p53(DO-1) followed by ECL using standard techniques.

Results: Forty compounds from the initial high through-put screen wereidentified as exhibiting inhibition of MDM2 auto-ubiquitination activityof more than 50% and further tested in all in vitro assays identifiedabove. Using the ARF peptide as a positive control for inhibition ofMDM2 ubiquitination, these assays identified four compounds that showedan ability to significantly inhibit MDM2 E3 ligase (FIG. 2A lanes 7, 9,10, 11). To determine whether the compounds were selective in theirability to inhibit MDM2, their effect on the activity of anotherunrelated E3 ligase, Nedd4 were tested. FIG. 2B shows typical resultsfrom that screen. Compound 98C07,10-(-3-chloro-phenyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-2,4-dione,shows some selectivity as an inhibitor of MDM2. By contrast, compound97H10,10-(4-chloro-phenyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-2,4-dioneinhibits all E3s and has been shown to block the activity of the E1enzyme, rather than the E3 (see 2D below). FIG. 2C is an independentexperiment further demonstrating the specificity of MDM2 by 98C07 (lane2). In FIG. 2D and FIG. 2E the effect of compounds on the more proximalsteps in the ubiquitination process, formation of thiol-ester linkageswith E1 (FIG. 2D) and with E2 (FIG. 2E). As is evident, while at leastone of the compounds inhibits more proximally, accounting for its lackof specificity, 98C07 inhibits neither thiol-ester linkages of ubiquitinwith E1 nor E2. FIG. 2F shows inhibition of MDM2 auto-ubiquitination bytwo other close family members of 98C07. These are 98D07,10-(-4-chloro-phenyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-2,4-dione,and 98E07,10-(-4-methyl-phenyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-2,4-dione.Two of these compounds have been evaluated for inhibition of p53, 98C07and 98D07 both exhibit significant dose-dependent inhibition of p53ubiquitination after being pre-bound to GST-MDM2 (FIG. 2G).

EXAMPLE 3 In vivo Cell Based Assay for Accumulation of MDM2 and p53

Methods: Normal human fibroblast MRC-5 were chosen to determine whetherthe compounds from the high throughput screening can inhibit the E3activity of Mdm2, i.e. stabilizing MDM2 and p53 in vivo. After beingseeded in 12-well tissue culture cluster overnight, the cells weretreated with 50 μM of the compounds for 8 hours. They were harvestedwith trypsin-EDTA, washed with PBS, and lysed with RIPA buffer.Following removal of insoluble pellet by centrifugation for 20 minutesat 10000 rpm, the lysate was separated on 4-20% gradientSDS-polyacrylamide gel and transferred onto nitrocellulose membrane. Themembrane was then blotted with anti-p53 antibody (DO-1), anti-MDM2antibodies (Ab-1 and Ab-2), anti-p27 (Santa Cruz), anti-Nedd4, andanti-p21Waf1 antibody. After extensive washing with PBS containing 0.5%Triton X-100, it was incubated with HRP-labeled donkey anti-mouseantibody and visualized using enhanced chemiluminescence.

Apoptosis and cell death were determined by FACS analysis of sub-G1nuclei or counting trypan blue-positive cells under microscope. Caspaseactivity was measured using fluorogenic substrate Ac-DEVD-AFC andCytoFluor multi-well plate reader (PerSeptive Biosystems).

Results: Compounds referred to as 98C, 98D, and 98E were all found tospecifically increase MDM2 and p53 compared to a series of othercompounds identified in the high throughput screen and were not furtherconsidered (FIG. 3A and FIG. 3B). As shown in FIG. 3B, they allincreased MDM2 and p53 levels in MRC-5 cells. The specificity of thecompounds was also revealed by examining whether they affect the levelsof the HECT domain E3 Nedd4 and p27, which is ubiquitinated by a RINGfinger-dependent SCF E3. As shown in FIG. 3B, the amounts of both Nedd4and p27 were not changed significantly by any of the 98 familycompounds. Moreover, while adriamycin increased only p53 and proteosomeinhibitor (LLNL) accumulated MDM2, p53, and p21, these compounds onlyincreased the amount of MDM2 and p53, but not p21, indicating they arespecific for the E3 activity of MDM2 (FIG. 3C).

To assess whether the increased p53 can induce apoptosis in transformedcells, use was made of the retinal epithelial cells (RPE), which areresistant to p53-mediated apoptosis, and matched E1A-transformed RPEcells, which have become sensitive to p53-mediated apoptosis. As withadriamycin and the proteosome inhibitor, all three compounds markedlyincreased apoptosis in E1A transformed cells, while little affect onnon-transformed RPE cells was observed (FIG. 4—left side). However,consistent with the predicted biological function of these compoundsthey all accumulated p53 in RPE cells regardless of whether they weretransformed with E1A (FIG. 4—right side). To evaluate whether apoptosisstimulated by these compounds requires p53 expression, E1A and Rastransformed MEFs from cells either expressing p53 (C8) or p53^(−/−) (A9)cells were compared in their capacity to activate effector caspases(FIG. 5 a) and cell death (FIG. 5 b).

Collectively, these results establish the capacity of these compounds toinhibit the ubiquitin ligase activity of MDM2 and the MDM2-mediatedubiquitination of p53. This is reflected in the stabilization of thesemolecules in cells and in relatively selective increased apoptosis intransformed cells over non-transformed cells and specifically intransformed cells that express p53.

EXAMPLE 4 Ubiquitin Ligase Method for GST-MDM-2 Fluorescent Detection

Glutathione sepharose beads (250 μl) are washed three times with 1 ml ofwash buffer (50 mM Tris pH 8.0, 2mM DTT, 5mM MgCl₂, 100 mM NaCl, 1%Triton-X100) buffer and about 1.5 ml of bacterial lysate containingGST-MDM2 or variants thereof, and candidate compounds are added to thebeads in separate vessels, e.g. a 1.5 ml eppendorf tube. The volumesused, are enough to conduct about 30 to about 100 reactions. The beadsand each of the compounds are cultured at about 4° C. for about an hourand are continuously tumbled so as to keep the beads from settling andallow for maximum adsorbance of the compounds to the beads. The nextsteps include centrifugation, aspiration of the supernatant and washingin buffer (50 mM Tris pH 8.0, 2 mM DTT, 5 mM MgCl₂, 100 mM NaCl, 1%Triton-X100). This is followed by resuspension in 25 μl of reactionbuffer (50 mM Tris pH 8.0, 2 mM DTT, 5 mM MgCl₂, 2 mM ATP) comprising 50ng of E1, 1-2 μl of E2 and 1 μg of His-6-Ubiquitin. The mixtures arethen incubated at 37° C. in a shaker at about 125 r.p.m. for up to about30 minutes. The reactions are stopped by diluting in 0.8 mls of coldwash buffer comprising PBS 0.05% Tween 20. The supernatant is aspiratedand India-horse radish peroxidase (500 μl of India-HRP 1:2000 diluted inwash buffer) is added and incubated for 15 minutes. After washing threetimes in wash buffer, 20 μl of each sample is assayed in 90 μl ofQuantablue (Pierce). The mixture is excited at 320 nm and emissions aremeasured, and compared to controls, including a control lacking E2.Increased fluorescence relative to a control indicates ubiquitination.

The foregoing description of the invention is merely illustrativethereof, and it is understood that variations and modifications can beeffected without departing from the spirit or scope of the invention asset forth in the following claims.

1. A method of treating cancer comprising administering to a subjectsuffering from cancer a compound of the following Formula IV

wherein R⁴ is selected from the group consisting of amino, halogen,hydroxy, C₂₋₆alkenyl, C₂₋₆alkynyl, C₁₋₆alkoxy, or mono ordi(C₁₋₆alkyl)amino; and pharmaceutically acceptable salts thereof andwherein the cells of said cancer retain a wild type p53 gene.
 2. Themethod of claim 1 wherein R⁴ is amino.
 3. The method of claim 1 whereinR⁴ is halogen.
 4. The method of claim 1 wherein R⁴ is hydroxyl.
 5. Themethod of claim 1 wherein R⁴ is C₂₋₆alkenyl or C₂₋₆alkynyl.
 6. Themethod of claim 1 wherein R⁴ is C₁₋₆alkoxy.
 7. The method of claim 1wherein R⁴ is mono or di(C₁₋₆alkyl)amino.
 8. The method of claim 1wherein R⁴ is selected from the group consisting of a chloro or fluorogroup, and R⁴ is attached to the 3 or 4 position of the phenyl ring. 9.The method of claim 1 wherein R⁴ is chloro and is attached to the 3 or 4position of the phenyl ring.
 10. The method of claim 1 wherein R⁴ isfluoro and is attached to the 3 or 4 position of the phenyl ring. 11.The method of claim 1 wherein R⁴ is methyl and is attached to the 3 or 4position of the phenyl ring.
 12. The method of claim 1 wherein thecompound is selected from the group consisting of10-(3-chloro-phenyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-2,4-dione, and10-(4-chloro-phenyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-2,4-dione; andpharmaceutically acceptable salts thereof.
 13. The method of claim 1wherein the subject has a solid tumor.
 14. The method of claim 1 whereinthe subject has a disseminated cancer.
 15. The method of claim 1 whereinthe patient is a mammal.
 16. The method of claim 1 wherein the patientis a primate or human.
 17. A method of treating cancer comprising:administering a compound of the following Formula IV to a subjectsuffering from cancer of the colon, prostate, breast or skin:

wherein R⁴ is selected from the group consisting of amino, halogen,hydroxy, C₂₋₆alkenyl, C₂₋₆alkynyl, C₁₋₆alkoxy, or mono ordi(C₁₋₆alkyl)amino; and pharmaceutically acceptable salts thereof andwherein the cells of said cancer retain a wild type p53 gene.
 18. Themethod of claim 17 wherein R⁴ is amino.
 19. The method of claim 17wherein R⁴ is halogen.
 20. The method of claim 17 wherein R⁴ ishydroxyl.
 21. The method of claim 17 wherein the compound is selectedfrom the group consisting of10-(3-chloro-phenyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-2,4-dione, and10-(4-chloro-phenyl)-7-nitro-10H-pyrimido[4,5-b]quinoline-2,4-dione; andpharmaceutically acceptable salts thereof.
 22. The method of claim 17wherein the subject is suffering from breast cancer.